Figure 6

5-Hydroxymethylcytosine (5-hmC) is dramatically enriched in both neuronal progenitors and differentiated post-mitotic cells of neuronal lineages. (A) 5-hmC Is visualised by immunochemistry in hippocampal A and ventricular (B) regions of mouse brain and cerebellar folia (C). Pyramidal layer (pl), dentate gyrus (dg), subventricular zone (svz) and corpus colossum (cc) are indicated. Scale bars are 250 μm. The experiments were performed using the Diagenode antibody. (D) Human neural stem cells (NSCs) exhibit high levels of 5-hmC staining, which does not disappear after 18 days of differentiation into neurons. hESC-derived NSCs (indicated) and neurons derived from them after 18 days (neurons 18 days) of differentiation were immunostained for 5-hmC. 5-hmC, DNA and phase views are indicated. Scale bars are 20 μm. The experiments were performed using the Diagenode antibody. (E) The upper panel shows a representative field of human NSC culture used for 5-hmC immunostaining in D stained for Sox1 (red nuclear signal), DAPI (blue) and nestin (green cytoplasmic signal). The majority of cells co-express both markers. Scale bar is 50 μm. The middle panel shows a representative field of the neuronal culture obtained after 18 days of differentiation and used for 5-hmC immunostaining in D, stained for β-III tubulin (green), Synapsin I (red in the colour image and presented as single channel in the grayscale image on the right) and DAPI (blue). The majority of cells co-express β-III tubulin and Synapsin (scale bar is 50 μm). The lower panel shows the same stained population at a higher magnification (scale bar is 10 μm). (F) The results of real-time RT-PCR analysis of Tet1/2/3 transcripts (indicated) in HDFs, human H1 ES cells and obtained from them human NSCs (indicated). The transcripts of all three Tet genes are highly expressed in human NSCs. The data were normalised with relative to GAPDH. Error is expressed as s.e.m.